2.1 Fcγ Receptors and their Role in the Immune System
The interaction of antibody-antigen complexes with cells of the immune system results in a wide array of responses, ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to immunomodulatory signals such as regulating lymphocyte proliferation and antibody secretion. All these interactions are initiated through the binding of the Fcγ domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses triggered by antibodies and immune complexes results from the structural heterogeneity of Fcγ receptors. Fcγ receptors share structurally related ligand binding domains which presumably mediate intracellular signaling.
The Fcγ receptors, members of the immunoglobulin gene superfamily of proteins, are surface glycoproteins that can bind the Fcγ portion of immunoglobulin molecules. Each member of the family recognizes immunoglobulins of one or more isotypes through a recognition domain on the a chain of the Fcγ receptor. Fcγ receptors are defined by their specificity for immunoglobulin subtypes. Fcγ receptors for IgG are referred to as FcγR, for IgE as FcεR, and for IgA as FcαR. Different accessory cells bear Fcγ receptors for antibodies of different isotype, and the isotype of the antibody determines which accessory cells will be engaged in a given response (reviewed by Ravetch J. V. et al. 1991, Annu. Rev. Immunol. 9: 457-92; Gerber J. S. et al. 2001 Microbes and Infection, 3: 131-139; Billadeau D. D. et al. 2002, The Journal of Clinical Investigation, 2(109): 161-1681; Ravetch J. V. et al. 2000, Science, 290: 84-89; Ravetch J. V. et al., 2001 Annu. Rev. Immunol. 19:275-90; Ravetch J. V. 1994, Cell, 78(4): 553-60). The different Fcγ receptors, the cells that express them, and their isotype specificity is summarized in Table 1 (adapted from Immunobiology: The Immune System in Health and Disease, 4th ed. 1999, Elsevier Science Ltd/Garland Publishing, New York).
Fcγ Receptors
Each member of this family is an integral membrane glycoprotein, possessing extracellular domains related to a C2-set of immunoglobulin-related domains, a single membrane spanning domain and an intracytoplasmic domain of variable length. There are three known FcγRs, designated FcγRI(CD64), FcγRII(CD32), and FcγIII(CD16). The three receptors are encoded by distinct genes; however, the extensive homology between the three family members suggest they arose from a common progenitor perhaps by gene duplication.
FcγRII(CD32)
FcγRII proteins are 40 KDa integral membrane glycoproteins which bind only the complexed IgG due to a low affinity for monomeric Ig (106 M−1). This receptor is the most widely expressed FcγR, present on all hematopoietic cells, including monocytes, macrophages, B cells, NK cells, neutrophils, mast cells, and platelets. FcγRII has only two immunoglobulin-like regions in its immunoglobulin binding chain and hence a much lower affinity for IgG than FcγRI. There are three human FcγRII genes (FcγRII-A, FcγII-B, FcγRII-C), all of which bind IgG in aggregates or immune complexes.
Distinct differences within the cytoplasmic domains of FcγRII-A and FcγRII-B create two functionally heterogenous responses to receptor ligation. The fundamental difference is that the A isoform initiates intracellular signaling leading to cell activation such as phagocytosis and respiratory burst, whereas the B isoform initiates inhibitory signals, e.g., inhibiting B-cell activation.
FcγRIII (CD16)
Due to heterogeneity within this class, the size of FcγRIII ranges between 40 and 80 KDa in mouse and man. Two human genes encode two transcripts, FcγRIIIA, an integral membrane glycoprotein, and FcγRIIIB, a glycosylphosphatidyl-inositol (GPI)-linked version. One murine gene encodes an FcγRIII homologous to the membrane spanning human FcγRIIIA. The FcγRIII shares structral characteristics with each of the other two FcγRs. Like FcγRII, FcγRIII binds IgG with low affinity and contains the corresponding two extracellular Ig-like domains. FcγRIIIA is expressed in macrophages, mast cells and is the lone FcγR in NK cells. The GPI-linked FcγRIIIB is currently known to be expressed only in human neutrophils.
Signaling through FcγRs
Both activating and inhibitory signals are transduced through the FcγRs following ligation. These diametrically opposing functions result from structural differences among the different receptor isoforms. Two distinct domains within the cytoplasmic signaling domains of the receptor called immunoreceptor tyrosine based activation motifs (ITAMs) or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account for the different responses. The recruitment of different cytoplasmic enzymes to these structures dictates the outcome of the FcγR-mediated cellular responses. ITAM-containing FcγR complexes include FcγRI, FcγRIIA, FcγIIIA, whereas ITIM-containing complexes only include FcγRIIB.
Human neutrophils express the FcγRIIA gene. FcγRIIA clustering via immune complexes or specific antibody cross-linking serves to aggregate ITAMs along with receptor-associated kinases which facilitate ITAM phosphorylation. ITAM phosphorylation serves as a docking site for Syk kinase, activation of which results in activation of downstream substrates (e.g., PI3K). Cellular activation leads to release of proinflammatory mediators.
The FcγRIIB gene is expressed on B lymphocytes; its extracellular domain is 96% identical to FcγRIIA and binds IgG complexes in an indistinguishable manner. The presence of an ITIM in the cytoplasmic domain of FcγRIIB defines this inhibitory subclass of FcγR. Recently the molecular basis of this inhibition was established. When co-ligated along with an activating FcγR, the ITIM in FcγRIIB becomes phosphorylated and attracts the SH2 domain of the inositol polyphosphate 5′-phosphatase (SHIP), which hydrolyzes phosphoinositol messengers released as a consequence of ITAM-containing FcγR-mediated tyrosine kinase activation, consequently preventing the influx of intracellular Ca++. Thus crosslinking of FcγRIIB dampens the activating response to FcγR ligation and inhibits cellular responsiveness. B cell activation, B cell proliferation and antibody secretion is thus aborted.
TABLE 1Receptors for the Fcγ Regions of Immunoglobulin IsotypesReceptorFcγRIFcγRII-AFcγRII-B2FcγRII-B1FcγRIIIFcγeRIFcγaRI(CD64)(CD32)(CD32)(CD32)(CD16)(CD89)BindingIgG1IgG1IgG1IgG1IgG1IgEIgA1, IgA2108 M−12 × 106 M−12 × 106 M−12 × 106 M−15 × 105 M−11010 M−1107 M−1CellMacrophagesMacrophagesMacrophagesB cellsNK cellsMast cellsMacrophagesTypeNeutrophilsNeutrophilsNeutrophilsMast cellsEosinophilEosinophilNeutrophilsEosinophilsEosinophilsEosinophilsMacrophagesBasophilsEosinophilsDendriticDendriticNeutrophilscellscellsMast CellsPlateletsLangerhancellsEffect ofUptakeUptakeUptakeNo uptakeInduction ofSecretionUptakeLigationStimulationGranuleInhibition ofInhibitionKillingofInduction ofActivation ofreleaseStimulationofgranuleskillingrespiratoryStimulationburstInduction ofkilling
2.2 Diseases of Relevance
2.2.1 Autoimmune Diseases
Autoimmune disease occurs when a specific adaptive immune response is mounted against self antigens. The normal consequence of an adaptive immune response against a foreign antigen is the clearance of the antigen from the body. Virus-infected cells, for example, are destroyed by cytotoxic T cells, whereas soluble antigens are cleared by formation of immune complexes of antibody and antigen, which are taken up by cells of the mononuclear phagocytic system such as macrophages. When an adaptive immune response develops against self antigens, however, it is usually impossible for immune effector mechanisms to eliminate the antigen completely, and so a sustained response occurs. The consequence is that the effector pathways of immunity cause chronic inflammatory injury to tissues, which may prove lethal. The mechanisms of tissue damage in autoimmune diseases are essentially the same as those that operate in protective immunity and in hypersensitivity diseases.
It is useful to distinguish two major patterns of autoimmune disease, the diseases in which the expression of autoimmunity is restricted to specific organs of the body, known as ‘organ-specific’ autoimmune diseases, and those in which many tissues of the body are affected, the ‘systemic’ autoimmune diseases. Examples of organ-specific autoimmune diseases are Hashimoto's thyroiditis and Graves' disease, each predominantly affecting the thyroid gland, and type I insulin-dependent diabetes mellitus (IDDM), which affects the pancreatic islets. Examples of systemic autoimmune disease are systemic lupus erythematosus (SLE) and primary Sjögren's syndrome, in which tissues as diverse as the skin, kidneys, and brain may all be affected.
Tissue injury in autoimmune disease results because the self antigen is an intrinsic component of the body and, consequently, the effector mechanisms of the immune system are directed at the body's own tissues. Also, because the adaptive immune response is incapable of removing the offending autoantigen from the body, the immune response persists, and there is a constant supply of new autoantigen, which amplifies the response.
IgG or IgM responses to antigens located on the surface of blood cells lead to the rapid destruction of these cells. An example of this is autoimmune hemolytic anemia, where antibodies against self antigens on red blood cells trigger destruction of the cells, leading to anemia. This can occur in two ways. Red cells with bound IgG or IgM antibody are rapidly cleared from the circulation by interaction with Fcγ or complement receptors, respectively, on cells of the fixed mononuclear phagocytic system; this occurs particularly in the spleen. Alternatively, the autoantibody-sensitized red cells are lysed by formation of the membrane-attack complex of complement. In autoimmune thrombocytopenic purpura, autoantibodies primarily against the GpIIb:IIIa fibrinogen receptor on platelets can cause thrombocytopenia (a depletion of platelets), which can in turn cause hemorrhage.
Current treatments for immunological disorders are nearly all empirical in origin, using immunosuppressive drugs identified by screening large numbers of natural and synthetic compounds. The drugs currently used to suppress the immune system can be divided into three categories: first, powerful anti-inflammatory drugs of the corticosteroid family such as prednisone; second, cytotoxic drugs such as azathioprine and cyclophosphamide; and third, fungal and bacterial derivatives, such as cyclosporin A, FK506 (tacrolimus), and rapamycin (sirolimus), which inhibit signaling events within T lymphocytes. These drugs are all very broad in their actions and inhibit protective functions of the immune system as well as harmful ones. Opportunistic infection is therefore a common complication of immuno-suppressive drug therapy. There thus still remains a need for developing safer, more effective therapeutic agents for autoimmune disorders.
Fcγ receptors have been implicated in the pathogenesis of autoimmune disorders. In particular, mice deficient in activating Fcγ receptors were unable to mount inflammatory responses when immunoglobulins (IgG) were bound to their cognate antigens (Sylvestere et al., 1994, Science, 265: 1095-8; Hazenbos et al., 1996, Immunity, 5: 181-8; Clynes et al., 1995, Immunity, 3: 21-26). In marked contrast, animals deficient in complement components had a normal inflammatory response to these experimentally induced cytotoxic antibodies and IgG-antigen complexes (Sylvestre et al., 1996, J. Exp. Med, 184: 2385-2392). This finding demonstrated that FcγRs provided the molecular coupling that allowed bound antibodies to elicit an effector cell response. This observation has led to a fundamental revision of the mechanism by which antibodies trigger inflammation as pathogenic agents in autoimmune diseases.
Idiopathic thrombocytopenic purpura (ITP), a disease in which the patient's immune system attacks and destroys platelets has been a key target for understanding the molecular basis of autoimmune disorders given the availability of experimental animal models (Bussel, 2000, Semin. Oncol. 27: 91-98). Antibodies to platelet glycoproteins have been implicated in the pathogenesis of ITP in humans (McMillan et al., 1981, N. Engl. J. Med., 304: 1135-1147). The development of a mouse model for this disease combined with the use of FcγR knockouts and transgenic mice has allowed greater insight into the mechanisms of pathogenesis and treatment. (NZW×BXSB) F1 mice spontaneously develop thrombocytopenia due to the production of autoantibodies (Oyaizu et al., 1988, J. Exp. Med., 167: 2017-22; Mizutani et al., 1993, Blood, 82: 837-844). An anti-platelet monoclonal antibody, 6A6, was derived from these mice and has been used in other mouse strains as a passive model for ITP. Clynes and Ravetch showed that in FcγR −/− mice, which are deficient in FcγRI and FcγRIIIA function, the monoclonal antibody 6A6 failed to induce thrombocytopenia (Clynes et al., 1995, Immunity, 3:21-26). Further studies demonstrated that the 6A6 antibody failed to induce platelet depletion in animals deleted for FcγRIII, but not in animals deleted for FcγRI. They further demonstrated that IVIG therapy was able to protect wild type animals, but not animals deleted for FcγRIIB, from platelet depletion. Wild type animals treated with IVIG showed increased expression of FcγRIIB (Samuelsson et al., 2001, Science, 291: 484-486). Thus these studies showed that IVIG acts not necessarily by the obvious mechanism of blocking the activating receptor but rather by inducing the inhibitory receptor, FcγRIIB.
Approximately 100,000 people in the United States have ITP including 18,000 with primary ITP, 50,000 with ITP secondary to HIV infection, and 30,000 with ITP secondary to other conditions. Among adults, about three times more women are affected than men, while in children the ratio is about even. The disease affects all age groups. There are approximately 20,000 new cases per year and estimates of incidence range broadly from about 10 to 125 per million people. Current therapeutic strategies to control ITP include administration of intravenous immunoglobulin (IVIG) or Anti-D (anti-rhesus globulin; which can also be delivered via rather than via IV infusion), immunosuppressive agents (such as steroids, azathioprine, or cyclosporine) or splenectomy. However, to date the therapeutic regimens for ITP are deficient and safer more efficacious treatment methods are needed.